Novel pH Selective, Highly Lytic Peptides Based on a Chimeric Influenza Hemagglutinin Peptide/Cell Penetrating Peptide Motif.

Delivery of macromolecular cargos such as siRNA to the cytosol after endocytosis remains a critical challenge. Numerous approaches including viruses, lipid nanoparticles, polymeric constructs, and various peptide-based approaches have yet to yield a general solution to this delivery issue. In this manuscript, we describe our efforts to design novel endosomolytic peptides that could be used to facilitate the release of cargos from a late endosomal compartment. These amphiphilic peptides, based on a chimeric influenza hemagglutinin peptide/cell-penetrating peptide (CPP) template, utilize a pH-triggering mechanism in which the peptides are protonated after acidification of the endosome, and thereby adopt an alpha-helical conformation. The helical forms of the peptides are lytically active, while the non-protonated forms are much less or non-lytically active at physiological pH. Starting from an initial lead peptide (INF7-Tat), we systematically modified the sequence of the chimeric peptides to obtain peptides with greatly enhanced lytic activity that maintain good pH selectivity in a red blood cell hemolysis assay.

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape.

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.

Rapid HATU-mediated solution phase siRNA conjugation.

Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.